FabH

ABSTRACT

The invention provides FabH polypeptides and DNA (RNA) encoding such FabH and a procedure for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing such FabH for the treatment of infection, particularly bacterial infections. Antagonists against such FabH and their use as a therapeutic to treat infections, particularly bacterial infections are also provided. Further provided are diagnostic assays for detecting diseases related to the presence of FabH nucleic acid sequences and the polypeptides in a host. Also provided are diagnostic assays for detecting polynucleotides encoding novel Fab family proteins and for detecting such polypeptides in a host.

This is a Divisional of application Ser. No. 08/746,797, filed Nov. 18,1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides; processes for making these polynucleotides and thesepolypeptides, and their variants; agonists and antagonists of thepolypeptides; and uses of these polynucleotides, polypeptides, variants,agonists and antagonists. In particular, in these and in other regards,the invention relates to polynucleotides and polypeptides of the Fab(fatty acid biosynthesis) family, hereinafter referred to as "FabH".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and Avery, Macleod and McCarty usingthis microbe. Despite the vast amount of research with S. pneumoniae,many questions concerning the virulence of this microbe remain. There isan umet medical need to employ Streptococcal genes and gene products astargets for the development of antibiotics.

The pathway for the biosynthesis of saturated fatty acids is verysimilar in prokaryotes and eukaryotes. However, whilst the chemicalreactions may not vary, the organisation of the biosynthetic apparatusis very different. Vertebrates and yeasts possess type I fatty acidsynthases (FASs) in which all of the enzymatic activities are encoded onone or two polypeptide chains, respectively. The acyl carrier protein(ACP) is an integral part of the complex. In contrast, in most bacterialand plant FASs (type II) each of the reactions are catalysed by distinctmonofunctional enzymes and the ACP is a discrete protein. Mycobacteriaare unique in that they possess both type I and II FASs; the former isinvolved in basic fatty acid biosynthesis whereas the latter is involvedin synthesis of complex cell envelope lipids such as mycolic acids.There therefore appears to be considerable potential for selectiveinhibition of the bacterial systems by broad spectrum antibacterialagents (Jackowski, S. 1992. In Emerging Targets in Antibacterial andAntifungal Chemotherapy. Ed. J. Sutcliffe & N. Georgopapadakou. Chapman& Hall, New York; Jackowski, S. et al. (1989). J. Biol. Chem. 264,7624-7629.)

The first step in the biosynthetic cycle is the condensation ofmalonyl-ACP with acetyl-CoA by FabH. In subsequent rounds malonyl-ACP iscondensed with the growing-chain acyl-ACP (FabB and FabF, synthases Iand II respectively). The second step in the elongation cycle isketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG).Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (either FabA orFabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACPby NADH-dependent enoyl-ACP reductase (FabI). Further rounds of thiscycle, adding two carbon atoms per cycle, eventually lead topalmitoyl-ACP whereupon the cycle is stopped largely due to feedbackinhibition of FabH and I by palmitoyl-ACP (Heath, et al, (1996), J.Biol. Chem. 271, 1833-1836). Fab H is therefore a major biosyntheticenzyme which is also a key regulatory point in the overall syntheticpathway (Heath, R. J. and Rock, C. O. 1996. J. Biol. Chem. 271,1833-1836; Heath, R. J. and Rock, C. O. 1996. J. Biol. Chem. 271,10996-11000).

The antibiotic thiolactomycin has broad-spectrum antibacterial activityboth in vivo and in vitro and has been shown to specifically inhibit allthree condensing enzymes. It is non-toxic and does not inhibit mammalianFASs (Hayashi, T. et al., 1984. J. Antibiotics 37, 1456-1461; Miyakawa,S. et al., 1982. J. Antibiotics 35, 411-419; Nawata, Y et al., 1989.Acta Cryst. C45, 978-979; Noto, T. et al., 1982. J. Antibiotics 35,401-410; Oishi, H. et al., 1982. J. Antibiotics 35, 391-396. Similarly,cerulenin is a potent inhibitor of FabB & F and is bactericidal but istoxic to eukaryotes because it competes for the fatty-acyl binding sitecommon to both FAS types (D'Agnolo, G. et al., 1973. Biochim. Biophys.Acta. 326, 155-166). Extensive work with these inhibitors has provedthat these enzymes are essential for viability. Little work has beencarried out in Gram-positive bacteria.

No mutants totally lacking FabH activity have been described.β-ketoacyl-ACP synthases are widely conserved enzymes in Gram-negativebacteria. For example E. coli FabH is 80% similar to the Haemophilusinfluenzae homolog.

No mammalian homologues to FabH have yet been identified. No marketedantibiotics are targeted against fatty acid biosynthesis, therefore itis unlikely that novel antibiotics would be rendered inactive by knownantibiotic resistance mechanisms. Moreover, this is a potentially broadspectrum target.

There is a clear unmet need for developing new classes of antibioticcompounds. Clearly, there is also a need for factors that may be used toscreen compounds for antibiotic activity, such as a simple highthrough-put assay for screening inhibitors of FAS. Such factors may alsobe used to determine their roles in pathogenesis of infection,dysfunction and disease. Identification and characterization of suchfactors, which can play a role in preventing, ameliorating or correctinginfections, dysfunctions or diseases are critical steps in makingimportant discoveries to improve human health.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelFabH peptides by homology between the amino acid sequence set out inFIG. 2 and known amino acid sequences of other proteins such asStreptococcus pyogenes FabH protein.

It is a further object of the invention, moreover, to providepolynucleotides that encode FabH polypeptides, particularlypolynucleotides that encode the polypeptide herein designated FabH.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding FabH polypeptides inthe sequence set out in FIG. 1 SEQ ID NO:1!, or a variant thereof.

In another particularly preferred embodiment of the present inventionthere is a novel protein from Streptococcus pneumoniae comprising theamino acid sequence of FIG. 2 SEQ ID NO:2!, or a variant thereof.

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the bacterial strain Streptococcus pneumoniae 0100993contained in NCIMB Deposit No. 40794.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding FabH, particularlyStreptococcus FabH, including mRNAs, cDNAs, genomic DNAs and, in furtherembodiments of this aspect of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising same.

In accordance with another aspect of the present invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of FabH andpolypeptides encoded thereby.

In accordance with this aspect of the invention there are provided novelpolypeptides of Streptococcus referred to herein as FabH as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprisingsame.

Among the particularly preferred embodiments of this aspect of theinvention are variants of FabH polypeptide encoded by naturallyoccurring alleles of the FabH gene.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned FabH polypeptides.

In accordance with yet another aspect of the present invention, thereare provided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia: assessing FabH expression; to treat, for example, otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, assaying genetic variation;and administering a FabH polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to FabH polynucleotide sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against FabH polypeptides.

In accordance with another aspect of the present invention, there areprovided FabH agonists which are also preferably bacteriostatic orbacteriocidal.

In accordance with yet another aspect of the present invention, thereare provided FabH antagonists which are also preferably bacteriostaticor bacteriocidal.

In a further aspect of the invention there are provided compositionscomprising a FabH polynucleotide or a FabH polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the polynucleotide sequence of Streptococcus pneumoniaeFabH SEQ ID NO:1!. The start codon of the coding sequence is underlinedand in bold text. The stop codon is underlined.

FIG. 2 shows the amino acid sequence of Streptococcus pneumoniae FabHSEQ ID NO:2! deduced from the polynucleotide sequence of FIG. 1.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe Examples. The explanations are provided as a convenience and are notlimitative of the invention.

FabH-BINDING MOLECULE, as used herein, refers to molecules or ions whichbind or interact specifically with FabH polypeptides or polynucleotidesof the present invention, including, for example enzyme substrates, cellmembrane components and classical receptors. Binding betweenpolypeptides of the invention and such molecules, including binding orbinding or interaction molecules may be exclusive to polypeptides of theinvention, which is preferred, or it may be highly specific forpolypeptides of the invention, which is also preferred, or it may behighly specific to a group of proteins that includes polypeptides of theinvention, which is preferred, or it may be specific to several groupsof proteins at least one of which includes a polypeptide of theinvention. Binding molecules also include antibodies andantibody-derived reagents that bind specifically to polypeptides of theinvention.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a polynucleotide region that regulatesreplication, transcription or translation or other processes importantto expression of the polypeptide in a host cell, or a polynucleotidecomprising both a region that encodes a polypeptide and a regionoperably linked thereto that regulates expression. Genetic elements maybe comprised within a vector that replicates as an episomal element;that is, as a molecule physically independent of the host cell genome.They may be comprised within plasmids. Genetic elements also may becomprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

HOST CELL is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences, which relationshipsare generally known as homology. In the art, identity also means thedegree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. Both identity and similarity can be readilycalculated (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). While there exist a number of methods to measure identity andsimilarity between two polynucleotide or two polypeptide sequences, bothterms are well known to skilled artisans (Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).Methods commonly employed to determine identity or similarity betweensequences include, but are not limited to those disclosed in Carillo,H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, GCG program package (Devereux, J., etal., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living organismin its natural state is not "isolated," but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis "isolated", as the term is employed herein.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are "polynucleotides" as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. Polynucleotidesembraces short polynucleotides often referred to as oligonucleotide(s).

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in the art. The term is used herein to refer to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds. As used herein, the term refers to both short chains,which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known, such as found in PROTEINS--STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993). Many detailed reviews are available on this subject, suchas, for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranstational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides are not always entirely linear. For instance,polypeptides may be generally as a result of posttranslational events,including natural processing event and events brought about by humanmanipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli or other cells, priorto proteolytic processing, almost invariably will be N-formylmethionine.During post-translational modification of the peptide, a methionineresidue at the NH₂ -terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionineless amino terminal variants of the protein of the invention.The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as, forexample, E coli. Accordingly, when glycosylation is desired, apolypeptide should be expressed in a glycosylating host, generally aeukaryotic cell. Insect cell often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to express efficiently mammalianproteins having native patterns of glycosylation, inter alia. Similarconsiderations apply to other modifications. It will be appreciated thatthe same type of modification may be present in the same or varyingdegree at several sites in a given polypeptide. Also, a givenpolypeptide may contain many types of modifications. In general, as usedherein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

VARIANT(S), as the term is used herein, are polynucleotides orpolypeptides that differ from a reference polynucleotide or polypeptiderespectively. Variants in this sense are described below and elsewherein the present disclosure in greater detail. (1) A polynucleotide thatdiffers in nucleotide sequence from another, reference polynucleotide.Generally, differences are limited so that the nucleotide sequences ofthe reference and the variant are closely similar overall and, in manyregions, identical. Changes in the nucleotide sequence of the variantmay be silent, i.e., they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference polypeptide. Changes in the nucleotidesequence of the variant may alter the amino acid sequence of apolypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below. (2) A polypeptide that differs in aminoacid sequence from another, reference polypeptide. Generally,differences are limited so that the sequences of the reference and thevariant are closely similar overall and, in many region, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination. (3) A variant mayalso be a fragment of a polynucleotide or polypeptide of the inventionthat differs from a reference polynucleotide or polypeptide sequence bybeing shorter than the reference sequence, such as by a terminal orinternal deletion. A variant of a polypeptide of the invention alsoincludes a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide, e.g., proproteins which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide. (4) A variant may also be (i) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. (5) A variant of thepolynucleotide or polypeptide may be a naturally occurring variant suchas a naturally occurring allelic variant, or it may be a variant that isnot known to occur naturally. Such non-naturally occurring variants ofthe polynucleotide may be made by mutagenesis techniques, includingthose applied to polynucleotides, cells or organisms, or may be made byrecombinant means. Among polynucleotide variants in this regard arevariants that differ from the aforementioned polynucleotides bynucleotide substitutions, deletions or additions. The substitutions,deletions or additions may involve one or more nucleotides. The variantsmay be altered in coding or non-coding regions or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Among polypeptide variantsin this regard are variants that differ from the aforementionedpolypeptides by amino acid substitutions, deletions or additions. Thesubstitutions, deletions or additions may involve one or more aminoacids. Alterations in the of the amino acids may be conservative ornon-conservative amino acid substitutions, deletions or additions. Allsuch variants defined above are deemed to be within the scope of thoseskilled in the art from the teachings herein and from the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates inter alia to novel FabH polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novel FabHgene of Streptococcus pneumoniae, which is related by amino acidsequence homology to Streptococcus pyogenes FabH polypeptide. Theinvention relates especially to FabH having the nucleotide and aminoacid sequences set out in FIG. 1 and FIG. 2 respectively, and to theFabH nucleotide and amino acid sequences of the DNA in NCIMB Deposit No.40794, which is herein referred to as "the deposited bacterial strain"or as the "DNA of the deposited bacterial strain."

Techniques are available to evaluate temporal gene expression inbacteria, particularly as it applies to viability under laboratory andhost infection conditions. A number of methods can be used to identifygenes which are essential to survival per se, or essential to theestablishment/maintenance of an infection. Identification of expressionof a sequence by one of these methods yields additional informationabout its function and permits the selection of such sequence forfurther development as a screening target. Briefly, these approachesinclude:

1) Signature Tagged Mutagenesis (STM)

This technique is described by Hensel et al., Science 269:400-403(1995), the contents of which is incorporated by reference forbackground purposes. Signature tagged mutagenesis identifies genesnecessary for the establishment/maintenance of infection in a giveninfection model.

The basis of the technique is the random mutagenesis of target organismby various means (e.g., transposons) such that unique DNA sequence tagsare inserted in close proximity to the site of mutation. The tags from amixed population of bacterial mutants and bacteria recovered from aninfected hosts are detected by amplification, radiolabeling andhybridization analysis. Mutants attenuated in virulence are revealed byabsence of the tag from the pool of bacteria recovered from infectedhosts.

In Streptococcus pneumoniae, because the transposon system is less welldeveloped, a more efficient way of creating the tagged mutants is to usethe insertion-duplication mutagenesis technique as described by Morrisonet al., J. Bacteriol. 159:870 (1984) the contents of which isincorporated by reference for background purposes.

2) In Vivo Expression Technology (IVET)

This technique is described by Camilli et al., Proc. Nat'l. Acad. Sci.USA. 91:2634-2638 (1994), the contents of which is incorporated byreference for background purposes. IVET identifies genes up-regulatedduring infection when compared to laboratory cultivation, implying animportant role in infection. Sequences identified by this technique areimplied to have a significant role in infectionestablishment/maintenance.

In this technique random chromosomal fragments of target organism arecloned upstream of a promoter-less reporter gene in a plasmid vector.The pool is introduced into a host and at various times after infectionbacteria may be recovered and assessed for the presence of reporter geneexpression. The chromosomal fragment carried upstream of an expressedreporter gene should carry a promoter or portion of a gene normallyupregulated during infection. Sequencing upstream of the reporter geneallows identification of the up regulated gene.

3) Differential display

This technique is described by Chuang et al., J. Bacteriol.175:2026-2036 (1993), the contents of which is incorporated by referencefor background purposes. This method identifies those genes which areexpressed in an organism by identifying mRNA present usingrandomly-primed RT-PCR. By comparing pre-infection and post infectionprofiles, genes up and down regulated during infection can be identifiedand the RT-PCR product sequenced and matched to library sequences.

4) Generation of conditional lethal mutants by transposon mutagenesis

This technique, described by de Lorenzo, V. et al., Gene 123:17-24(1993); Neuwald, A. F. et al., Gene 125: 69-73(1993); and Takiff, H. E.et al., J. Bacteriol. 174:1544-1553(1992), the contents of which isincorporated by reference for background purposes, identifies geneswhose expression are essential for cell viability.

In this technique transposons carrying controllable promoters, whichprovide transcription outward from the transposon in one or bothdirections, are generated. Random insertion of these transposons intotarget organisms and subsequent isolation of insertion mutants in thepresence of inducer of promoter activity ensures that insertions whichseparate promoter from coding region of a gene whose expression isessential for cell viability will be recovered. Subsequent replicaplating in the absence of inducer identifies such insertions, since theyfail to survive. Sequencing of the flanking regions of the transposonallows identification of site of insertion and identification of thegene disrupted. Close monitoring of the changes in cellularprocesses/morphology during growth in the absence of inducer yieldsinformation on likely function of the gene. Such monitoring couldinclude flow cytometry (cell division, lysis, redox potential, DNAreplication), incorporation of radiochemically labeled precursors intoDNA, RNA, protein, lipid, peptidoglycan, monitoring reporter enzyme genefusions which respond to known cellular stresses.

5) Generation of conditional lethal mutants by chemical mutagenesis

This technique is described by Beckwith, J., Methods in Enzymology 204:3-18(1991), the contents of which are incorporated herein by referencefor background purposes. In this technique random chemical mutagenesisof target organism, growth at temperature other than physiologicaltemperature (permissive temperature) and subsequent replica plating andgrowth at different temperature (e.g., 42° C. to identify ts, 25° C. toidentify cs) are used to identify those isolates which now fail to grow(conditional mutants). As above close monitoring of the changes upongrowth at the non-permissive temperature yields information on thefunction of the mutated gene. Complementation of conditional lethalmutation by library from target organism and sequencing of complementinggene allows matching with library sequences.

Each of these techniques may have advantages or disadvantage dependingon the particular application. The skilled artisan would choose theapproach that is the most relevant with the particular end use in mind.For example, some genes might be recognised as essential for infectionbut in reality are only necessary for the initiation of infection and sotheir products would represent relatively unattractive targets forantibacterials developed to cure established and chronic infections.

6) RT-PCR

Bacterial messenger RNA, preferably that of Streptococcus pneumoniae, isisolated from bacterial infected tissue e.g. 48 hour murine lunginfections, and the amount of each mRNA species assessed by reversetranscription of the RNA sample primed with random hexanucleotidesfollowed by PCR with gene specific primer pairs. The determination ofthe presence and amount of a particular mRNA species by quantificationof the resultant PCR product provides information on the bacterial geneswhich are transcribed in the infected tissue. Analysis of genetranscription can be carried out at different times of infection to gaina detailed knowledge of gene regulation in bacterial pathogenesisallowing for a clearer understanding of which gene products representtargets for screens for novel antibacterials. Because of the genespecific nature of the PCR primers employed it should be understood thatthe bacterial mRNA preparation need not be free of mammalian RNA. Thisallows the investigator to carry out a simple and quick RNA preparationfrom infected tissue to obtain bacterial mRNA species which are veryshort lived in the bacterium (in the order of 2 minute halflives).Optimally the bacterial mRNA is prepared from infected murine lungtissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL)for very short periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of bacterial 16S ribosomal RNA,preferably that of Streptococcus pneumoniae, as detected by probingNortherns with a suitably labeled sequence specific oligonucleotideprobe. Typically a 5' dye labelled primer is used in each PCR primerpair in a PCR reaction which is terminated optimally between 8 and 25cycles. The PCR products are separated on 6% polyacrylamide gels withdetection and quantification using GeneScanner (manufactured by ABI).

Use of the of these technologies when applied to the sequences of thepresent invention enables identification of bacterial proteins expressedduring infection, inhibitors of which would have utility inanti-bacterial therapy.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the FabH polypeptidehaving the deduced amino acid sequence of FIG. 2 SEQ ID NO:2!.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 SEQ ID NO:1!, a polynucleotide of the presentinvention encoding FabH polypeptide may be obtained using standardcloning and screening, such as those for cloning and sequencingchromosomal DNA fragments from Streptococcus pneumoniae 0100993 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide of the invention sequence, such asthat sequence given in FIG. 1 SEQ ID NO:1! typically a library of clonesof chromosomal DNA of Streptococcus pneumoniae 0100993 in E. coli orsome other suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing high stringency washes. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see Screening By Hybridization 1.90 and Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Illustrative of the invention, thepolynucleotide set out in FIG. 1 SEQ ID NO:1! was discovered in a DNAlibrary derived from Streptococcus pneumoniae 0100993.

FabH of the invention is structurally related to other proteins of theFab family, as shown by the results of sequencing the DNA encoding FabHof the deposited bacterial strain. The DNA sequence thus obtained is setout in FIG. 1 SEQ ID NO:1!. It contains an open reading frame encoding aprotein of having about the number of amino acid residues set forth inFIG. 2 SEQ ID NO:2! with a deduced molecular weight that can becalculated using amino acid residue molecular weight values well knownin the art. The protein exhibits greatest homology to Streptococcuspyogenes FabH protein among known proteins. FabH of FIG. 2 SEQ ID NO:2!has about 61% identity over its entire length and about 74% similarityover its entire length with the amino acid sequence of Streptococcuspyogenes FabH polypeptide.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1 SEQ ID NO:1!.It also may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of FIG. 2 SEQ ID NO:2!.

Polynucleotides of the present invention which encode the polypeptide ofFIG. 2 SEQ ID NO:2! may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to non-coding 5' and 3' sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals, for example), ribosome binding, mRNA stabilityelements, and additional coding sequence which encode additional aminoacids, such as those which provide additional functionalities. Thus, forinstance. the polypeptide may be fused to a marker sequence, such as apeptide, which facilitates purification of the fused polypeptide. Incertain embodiments of this aspect of the invention, the marker sequenceis a hexa-histidine peptide, such as the tag provided in the pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Nat. Acad. Sci., USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag may also be used tocreate fusion proteins and corresponds to an epitope derived ofinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984), for instance. Polynucleotides of the inventionalso include, but are not limited to, polynucleotides comprising astructural gene and its naturally associated genetic elements.

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlybacterial, and more particularly the Streptococcus pneumoniae FabHhaving the amino acid sequence set out in FIG. 2 SEQ ID NO:2!. The termencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for variants of the polypeptidehaving the deduced amino acid sequence of FIG. 2 SEQ ID NO:2!.

Among particularly preferred embodiments of the invention arepolynucleotides encoding polypeptides having the amino acid sequence ofFabH set out in FIG. 2 SEQ ID NO:2!, and variants thereof.

Further particularly preferred embodiments are polynucleotides encodingFabH variants, which have the amino acid sequence of FabH polypeptide ofFIG. 2 SEQ ID NO:2! in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2,1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of FabH.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding FabH polypeptide having the amino acid sequence set out in FIG.2 SEQ ID NO:2!, and polynucleotides which are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover their entire length to a polynucleotide encoding FabH polypeptideof the Streptococcus pneumoniae DNA of the deposited bacterial strainand polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments in this respect, moreover, are polynucleotideswhich encode polypeptides which retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the DNA ofFIG. 1 SEQ ID NO:1!.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding FabH and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the FabH gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less. An example of a probeuseful to detect the polynucleotide of the invention is5'-TAAGGCAGTCGATTGGTCGG-3' SEQ ID NO.3!.

For example, the coding region of the FabH gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofcDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsof and diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to polynucleotide assays, inter alia.

The polynucleotides of the invention that are oligonucleotides,including SEQ ID NOS:3 and 4, derived from the sequences of SEQ ID NOS:1and 2 may be used in the processes herein as described, but preferablyfor PCR, to determine whether or not the Streptococcus pneumoniae genesidentified herein in whole or in part are transcribed in infectedtissue. It is recognized that such sequences will also have utility indiagnosis of the stage of infection and type of infection the pathogenhas attained.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited materials

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

A deposit containing a Streptococcus pneumoniae bacterial strain hasbeen deposited with the National Collections of Industrial and MarineBacteria Ltd. (NCIMB), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotlandon 11 Apr. 1996 and assigned NCIMB Deposit No. 40794. The Streptococcuspneumoniae bacterial strain deposit is referred to herein as "thedeposited bacterial strain" or as "the DNA of the deposited bacterialstrain."

The deposited material is a bacterial strain that contains the fulllength FabH DNA, referred to as "NCIMB 40794" upon deposit.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a FabH polypeptide which has adeduced amino acid sequence of 324 amino acids in length, as set forthin FIG. 2 SEQ ID NO:2!, and has a deduced molecular weight of 34.902kilodaltons.

The invention also relates to variants of these polypeptides. Among theparticularly preferred embodiments of the invention in this regard arepolypeptides having the amino acid sequence of FabH set out in FIG. 2SEQ ID NO:2!, and variants thereof. Other particularly preferredembodiments of the invention are polypeptides having the amino acidsequence of the FabH, and variants thereof.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants having theamino acid sequence of the FabH polypeptide of FIG. 2 SEQ ID NO:2!, inwhich several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acidresidues are substituted, deleted or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the FabH.Also especially preferred in this regard are conservative substitutions.Most highly preferred are polypeptides having the amino acid sequence ofFIG. 2 SEQ ID NO:2! without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide ofFIG. 2 SEQ ID NO:2! (in particular the mature polypeptide) as well aspolypeptides which have at least 70% identity to the polypeptide of FIG.2 SEQ ID NO:2!, preferably at least 80% identity to the polypeptide ofFIG. 2 SEQ ID NO:2!, and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of FIG. 2 SEQ IDNO:2! and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of FIG. 2 SEQ IDNO:2! and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

Variants that are fragments of the polypeptides of the present inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides. Variants thatare fragments of the polynucleotides of the present invention may beused to synthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising variants that are fragments of FabH, mostparticularly fragments of FabH having the amino acid set out in FIG. 2SEQ ID NO:2!, and variants of the FabH of FIG. 2 SEQ ID NO:2!.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned FabH polypeptides and variants thereof.

Such fragments may be "free-standing," i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a FabH polypeptideof the present comprised within a precursor polypeptide designed forexpression in a host and having heterologous pre and pro-polypeptideregions fused to the amino terminus of the FabH fragment and anadditional region fused to the carboxyl terminus of the fragment.Therefore, fragments in one aspect of the meaning intended herein,refers to the portion or portions of a fusion polypeptide or fusionprotein derived from FabH.

Representative examples of polypeptide fragments of the invention,include, for example, fragments from amino acid number 1-20, 21-40,41-60, 61-80, 81-100, and 101-120, 121-140, 141-160, 161-180, 181-200,201-220, 221-240, 241-260, 261-280, 281-300, 301-324, and anycombination of these amino acid fragments.

In this context "about" herein includes the particularly recited rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

Preferred fragments of the invention include, for example, truncationpolypeptides of FabH. Truncation polypeptides include FabH polypeptideshaving the amino acid sequence of FIG. 2, or of variants thereof, exceptfor deletion of a continuous series of residues (that is, a continuousregion, part or portion) that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus or, asin double truncation mutants, deletion of two continuous series ofresidues, one including the amino terminus and one including thecarboxyl terminus. Fragments having the size ranges set out about alsoare preferred embodiments of truncation fragments, which are especiallypreferred among fragments generally. Degradation forms of thepolypeptides of the invention in a host cell, particularly aStreptococcus, are also preferred.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of FabH. Preferredembodiments of the invention in this regard include fragments thatcomprise alpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions of FabH, and combinations of such fragments.

Preferred regions are those that mediate activities of FabH. Most highlypreferred in this regard are fragments that have a chemical, biologicalor other activity of FabH, including those with a similar activity or animproved activity, or with a decreased undesirable activity. Furtherpreferred polypeptide fragments are those that are antigenic orimmunogenic in an animal, especially in a human.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespond to the preferred fragments, as discussed above.

Vectors, host cells, expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. Introduction of apolynucleotides into the host cell can be affected by calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

Polynucelotide constructs in host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Plasmids generallyare designated herein by a lower case p preceded and/or followed bycapital letters and/or numbers, in accordance with standard namingconventions that are familiar to those of skill in the art. Startingplasmids disclosed herein are either commercially available, publiclyavailable, or can be constructed from available plasmids by routineapplication of well known, published procedures. Many plasmids and othercloning and expression vectors that can be used in accordance with thepresent invention are well known and readily available to those of skillin the art.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques, such as, for example,those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude, but are not limited to, the phage lambda PL promoter, the E.coli lac, trp and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating codon, for example, AUG or GUG, at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as transcription factors, repressor binding sitesand termination, among others.

Vectors for propagation and expression generally will include selectablemarkers and amplification regions, such as, for example, those set forthin Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia, and pBR322 (ATCC 37017). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. These vectors are listed solely by way of illustration of themany commercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase ("CAT") transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable, such as pKK232-8 and pCM7. Promoters for expression ofpolynucleotides of the present invention include not only well known andreadily available promoters, but also promoters that readily may beobtained by the foregoing technique, using a reporter gene.

Among known prokaryotic promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous sarcoma virus ("RSV"), and metallothionein promoters, suchas the mouse metallothionein-I promoter.

Recombinant expression vectors will include, for example, origins ofreplication, a promoter preferably derived from a highly-expressed geneto direct transcription of a downstream structural sequence, and aselectable marker to permit isolation of vector containing cells afterexposure to the vector.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5' to aribosome binding site. The ribosome binding site will be 5' to the codonthat initiates translation of the polypeptide to be expressed, forexample AUG or GUG. Generally, there will be no other open readingframes that begin with an initiation codon, usually AUG, and lie betweenthe ribosome binding site and the initiation codon. Also, generally,there will be a translation stop codon at the end of the polypeptide andthere will be a polyadenylation signal in constructs for use ineukaryotic hosts. Transcription termination signal appropriatelydisposed at the 3' end of the transcribed region may also be included inthe polynucleotide construct.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunolglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another proteinor part thereof. In drug discovery, for example, proteins have beenfused with antibody Fc portions for the purpose of high-throughputscreening assays to identify antagonists. See, D. Bennett et al.,Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson etal., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471(1995).

Mammalian expression vectors may comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation regions, splice donor and acceptor sites,transcriptional termination sequences, and 5' flanking non-transcribedsequences that are necessary for expression.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

FabH polypeptide can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingprotein may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Polynucleotide assays

This invention is also related to the use of the FabH polynucleotides todetect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of FabH in a eukaryote, particularly amammal, and especially a human, will provide a diagnostic method fordiagnosis of a disease. Eukaryotes (herein also "individual(s)"),particularly mammals, and especially humans, infected with an organismcomprising the FabH gene may be detected at the DNA level by a varietyof techniques. Nucleic acids for diagnosis may be obtained from aninfected individual's cells and tissues, such as bone, blood, muscle,cartilage, and skin. Genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986) prior to analysis. RNA or cDNA may also be used in thesame ways. As an example, PCR primers complementary to the nucleic acidencoding FabH can be used to identify and analyze FabH presence and/orexpression. Using PCR, characterization of the strain of prokaryotepresent in a eukaryote, particularly a mammal, and especially a human,may be made by an analysis of the genotype of the prokaryote gene. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the genotype of a referencesequence. Point mutations can be identified by hybridizing amplified DNAto radiolabeled FabH RNA or alternatively, radiolabeled FabH antisenseDNA sequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic characterization based on DNA sequence differences may beachieved by detection of alteration in electrophoretic mobility of DNAfragments in gels, with or without denaturing agents. Small sequencedeletions and insertions can be visualized by high resolution gelelectrophoresis. DNA fragments of different sequences may bedistinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science, 230: 1242(1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Nat. Acad. Sci., USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Cells carrying mutations or polymorphisms in the gene of the presentinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to the nucleicacid encoding FabH can be used to identify and analyze mutations.Examples of representative primers SEQ ID NOS:4, 5, 6 and 7! are shownbelow in Table 1.

                  TABLE 1                                                         ______________________________________                                        Primers used for amplification of FabH polynucleotides                        SEQ ID NO                                                                              PRIMER SEQUENCE                                                      ______________________________________                                        4        5'-ATGGCTTTTGCAAAAATAAGTCAGGTTGC-3'                                  5        5'-ACAATGTGTTCACCACATGATTAC-3'                                       6        5'-TAAGGCAGTCGATTGGTCGG-3'                                           7        5'-GACTGTGCGTCCATCCATCT-3'                                           ______________________________________                                    

These primers may be used for amplifying FabH DNA isolated from a samplederived from an individual. For amplifying FabH DNA, it is preferredthat the primer sequences of SEQ ID NO:4 and 5 are used as a pair andthat the primers sequences of SEQ ID NO:6 and 7 are used as a pair. Theinvention also provides the primers of Table 1 with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. The primers may beused to amplify the gene isolated from the individual such that the genemay then be subject to various techniques for elucidation of the DNAsequence. In this way, mutations in the DNA sequence may be diagnosed.

The invention provides a process for diagnosing, disease, preferablybacterial infections, more preferably Streptococcus pneumoniae, and mostpreferably otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid, comprising determining from a sample derived from an individual aincreased level of expression of polynucleotide having the sequence ofFIG. 1 SEQ ID NO: 1!. Increased expression of FabH polynucleotide can bemeasured using any on of the methods well known in the art for thequantation of polynucleotides, such as, for example, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

Polypeptide assays

The present invention also relates to a diagnostic assays such asquantitative and qualitative assays for detecting levels of FabH proteinin cells and tissues, including determination of normal and abnormallevels. Thus, for instance, a diagnostic assay in accordance with theinvention for detecting over-expression of FabH protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a FabH protein, in a sample derived from a host are well-knownto those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays. Among these ELISAs frequently are preferred. An ELISAassay initially comprises preparing an antibody specific to FabH,preferably a monoclonal antibody. In addition a reporter antibodygenerally is prepared which binds to the monoclonal antibody. Thereporter antibody is attached a detectable reagent such as radioactive,fluorescent or enzymatic reagent, in this example horseradish peroxidaseenzyme.

Antibodies

The polypeptides and variants thereof, or cells expressing them can beused as an immunogen to produce antibodies thereto. The presentinvention includes, for examples monoclonal and polyclonal antibodies,chimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique known in the artwhich provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

Alternatively phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, antibodies against FabH may be employed to inhibitand/or treat infections, particularly bacterial infections andespecially otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants which form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognised by certain antibodies which, when raised to theprotein or polypeptide according to the present invention, interferewith the immediate physical interaction between pathogen and mammalianhost. The term "immunologically equivalent derivative" as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between pathogen andmammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem1989:264,16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS, 1986:83,9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS 1984:81,5849).

FabH-binding molecules and assays

This invention also provides a method for identification of molecules,such as binding molecules, that bind FabH. Genes encoding proteins thatbind FabH, can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting. Such methodsare described in the art, such as in Coligan et al., Current Protocolsin Immunology 1(2): Chapter 5 (1991). Also, a labeled ligand can bephotoaffinity linked to a cell extract. Polypeptides of the inventionalso can be used to assess FabH binding capacity of FabH-bindingmolecules, in cells or in cell-free preparations.

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics.

Antagonists and agonists--assays and molecules

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of FabHpolypeptides or polynucleotides, such as its interaction withFabH-binding molecules.

For example, to screen for agonists or antagoists, a synthetic reactionmix, a cellular compartment, such as a membrane, cell envelope or cellwall, or a preparation of any thereof, may be prepared from a cell thatexpresses a molecule that binds FabH. The preparation is incubated withlabeled FabH in the absence or the presence of a candidate moleculewhich may be a FabH agonist or antagonist. The ability of the candidatemolecule to bind the binding molecule is reflected in decreased bindingof the labeled ligand. Molecules which bind gratuitously, i.e., withoutinducing the effects of FabH on binding the FabH binding molecule, aremost likely to be good antagonists. Molecules that bind well and eliciteffects that are the same as or closely related to FabH are agonists.

FabH-like effects of potential agonists and antagonists may by measured,for instance, by determining activity of a reporter system followinginteraction of the candidate molecule with a cell or appropriate cellpreparation, and comparing the effect with that of FabH or moleculesthat elicit the same effects as FabH. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in FabH activity, and binding assays known in theart.

Another example of an assay for FabH antagonists is a competitive assaythat combines FabH and a potential antagonist with membrane-boundFabH-binding molecules, recombinant FabH binding molecules, naturalsubstrates or ligands, or substrate or ligand mimetics, underappropriate conditions for a competitive inhibition assay. FabH can belabeled, such as by radioactivity or a colorimetric compound, such thatthe number of FabH molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducingFabH-induced activities, thereby preventing the action of FabH byexcluding FabH from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of FabH.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the initial physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used: i) in the prevention of adhesion ofbacteria, in particular gram positive bacteria, to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; ii) to block FabH protein mediated mammaliancell invasion by, for example, initiating phosphorylation of mammaliantyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211 (1992); iii)to block bacterial adhesion between mammalian extracellular matrixproteins and bacterial FabH proteins which mediate tissue damage; iv) toblock the normal progression of pathogenesis in infections initiatedother than by the implantation of in-dwelling devices or by othersurgical techniques.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein uponexpression can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The antagonists and agonists may be employed for instance to inhibitotitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with FabH, or a fragment or variantthereof, adequate to produce antibody to protect said individual frominfection, particularly bacterial infection and most particularlyStreptococcus infections. Yet another aspect of the invention relates toa method of inducing immunological response in an individual whichcomprises, through gene therapy, delivering gene encoding FabH, or afragment or a variant thereof, for expressing FabH, or a fragment or avariant thereof in vivo in order to induce an immunological response toproduce antibody to protect said individual from disease.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable or having inducedwithin it an immunological response, induces an immunological responsein such host to a FabH or protein coded therefrom, wherein thecomposition comprises a recombinant FabH or protein coded therefromcomprising DNA which codes for and expresses an antigen of said FabH orprotein coded therefrom.

The FabH or a fragment thereof may be fused with co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. Thus fused recombinant protein, preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of Streptococcus pneumoniae infectionin mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused e.g. by mechanical, chemical or thermal damage or by implantationof indwelling devices, or wounds in the mucous membranes, such as themouth, mammary glands, urethra or vagina.

The present invention also includes a vaccine formulation whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain FabH,it is to be understood that this covers fragments of the naturallyoccurring protein and similar proteins with additions, deletions orsubstitutions which do not substantially affect the immunogenicproperties of the recombinant protein.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent Streptococcus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response.

A suitable unit dose for vaccination is 0.5-5 μg/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks.

With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of bacteria containing the FabH protein.

In order to facilitate understanding of the following example certainfrequently occurring methods and/or terms will be described.

EXAMPLES

The present invention is further described by the following examples.These exemplification's, while illustrating certain specific aspects ofthe invention, do not portray the limitations or circumscribe the scopeof the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Example 1

Library Production

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. In some cases the sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO:1. Librariesmay be prepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.Amplification can be performed using the primers in Table 1 SEQ ID NOS:3and 4!.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

FabD enzyme activity analysis

The activity of FabH can be measured using ¹⁴ C -acetylCoA as substrateand TCA-soluble malonyl-ACP as acceptor (Bruck, F. M. et al., 1996.Planta 198, 271-278; Jackowski, S. et al. (1989). J. Biol. Chem. 264,7624-7629; Heath, R. J. and Rock, C. O. 1996. J. Biol. Chem. 271,10996-11000) and measuring the incorporation of ¹⁴ C into insolubleacetoacetyl-ACP. Thiolactomycin can be used as a positive control andtest compounds may be added to this assay to determine whether theyagonise or antagonise enzyme activity.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 975 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGGCTTTTGCAAAAATAAGTCAGGTTGCTCATTATGTGCCAGAGCAAGTGGTTACAAAT60                CACGACTTGGCTCAGATTATGGATACCAATGATGAGTGGATTTCAAGTCGAACGGGAATA120               CGACAAAGGCATATTTCAAGAACAGAATCTACCAGTGATTTGGCTACAGAGGTTGCTAAG180               AAACTGATGGCAAAAGCTGGAATAACAGGAAAAGAACTGGATTTTATCATCCTAGCTACC240               ATTACTCCAGATTCGATGATGCCCTCTACAGCTGCTCGTGTTCAAGCTAATATTGGCGCT300               AATAAAGCCTTTGCTTTTGACTTAACCGCGGCTTGCAGTGGATTTGTATTTGCTCTTTCA360               ACTGCTGAAAAGTTTATCGCTTCTGGTCGCTTTCAAAAAGGCTTGGTGATTGGTAGTGAA420               ACCCTCTCTAAGGCAGTCGATTGGTCGGATCGATCAACAGCTGTGTTGTTTGGAGATGGT480               GCTGGTGGTGTCTTGTTAGAAGCTAGCGAGCAAGAGCATTTCTTAGCTGAGAGTCTTAAT540               AGCGATGGAAGTCGCAGCGAGTGTTTAACTTATGGGCATTCAGGTTTGCATTCTCCATTT600               TCAGATCAAGAAAGTGCAGATTCGTTTTTGAAGATGGATGGACGCACAGTCTTTGATTTT660               GCCATTCGAGATGTAGCCAAGTCTATCAAGCAGACTATTGATGAATCTCCTATAGAGGTG720               ACAGACTTGGATTATCTGCTACTTCATCAAGCCAATGACCGTATTTTGGATAAGATGGCT780               AGAAAAATTGGTGTTGACCGAGCCAAACTTCCAGCCAATATGATGGAATATGGCAATACC840               AGTGCAGCCAGTATCCCGATTTTACTTTCAGAGTGTGTAGAACAAGGTCTCATCCCTTTA900               GATGGTAGCCAGACTGTTCTTCTATCAGGCTTCGGTGGAGGCTTGACCTGGGGCACGCTC960               ATTCTTACAATTTAG975                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 324 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaPheAlaLysIleSerGlnValAlaHisTyrValProGluGln                              151015                                                                        ValValThrAsnHisAspLeuAlaGlnIleMetAspThrAsnAspGlu                              202530                                                                        TrpIleSerSerArgThrGlyIleArgGlnArgHisIleSerArgThr                              354045                                                                        GluSerThrSerAspLeuAlaThrGluValAlaLysLysLeuMetAla                              505560                                                                        LysAlaGlyIleThrGlyLysGluLeuAspPheIleIleLeuAlaThr                              65707580                                                                      IleThrProAspSerMetMetProSerThrAlaAlaArgValGlnAla                              859095                                                                        AsnIleGlyAlaAsnLysAlaPheAlaPheAspLeuThrAlaAlaCys                              100105110                                                                     SerGlyPheValPheAlaLeuSerThrAlaGluLysPheIleAlaSer                              115120125                                                                     GlyArgPheGlnLysGlyLeuValIleGlySerGluThrLeuSerLys                              130135140                                                                     AlaValAspTrpSerAspArgSerThrAlaValLeuPheGlyAspGly                              145150155160                                                                  AlaGlyGlyValLeuLeuGluAlaSerGluGlnGluHisPheLeuAla                              165170175                                                                     GluSerLeuAsnSerAspGlySerArgSerGluCysLeuThrTyrGly                              180185190                                                                     HisSerGlyLeuHisSerProPheSerAspGlnGluSerAlaAspSer                              195200205                                                                     PheLeuLysMetAspGlyArgThrValPheAspPheAlaIleArgAsp                              210215220                                                                     ValAlaLysSerIleLysGlnThrIleAspGluSerProIleGluVal                              225230235240                                                                  ThrAspLeuAspTyrLeuLeuLeuHisGlnAlaAsnAspArgIleLeu                              245250255                                                                     AspLysMetAlaArgLysIleGlyValAspArgAlaLysLeuProAla                              260265270                                                                     AsnMetMetGluTyrGlyAsnThrSerAlaAlaSerIleProIleLeu                              275280285                                                                     LeuSerGluCysValGluGlnGlyLeuIleProLeuAspGlySerGln                              290295300                                                                     ThrValLeuLeuSerGlyPheGlyGlyGlyLeuThrTrpGlyThrLeu                              305310315320                                                                  IleLeuThrIle                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TAAGGCAGTCGATTGGTCGG20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGGCTTTTGCAAAAATAAGTCAGGTTGC29                                               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ACAATGTGTTCACCACATGATTAC24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TAAGGCAGTCGATTGGTCGG20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GACTGTGCGTCCATCCATCT20                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated FabH polypeptide consisting of anamino acid sequence which is at least 70% identical to amino acid 1 to324 of SEQ ID NO:2.
 2. An isolated polypeptide consisting of an aminoacid sequence as set forth in SEQ ID NO:2.